Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial nitric oxide synthase (eNOS). Diminished NO formation and subsequent impaired relaxation have been proposed to be due to reduced endothelial BH4 levels, which enhances superoxide formation from eNOS. This idea gained support from electron paramagnetic resonance spin trapping studies showing that fully reduced BH4 inhibits superoxide release form eNOS while oxidized BH4 has no effect. In addition, these studies demonstrated that saturation of BH4- binding site is critical for inhibiting superoxide and that displacement of the cofactor from eNOS by oxidized BH4 analogs such as sepiapterin and 7,8-dihydrobiopterin elevates superoxide production. Based on these findings we hypothesized that the ratio between fully reduced and oxidized BH4 regulates superoxide generation from endothelial nitric oxide synthase. Previous studies used L-arginine and L-arginine analogs to implicate eNOS as a source of superoxide. However, spin trapping studies showed that these compounds have little effect on superoxide release from purified eNOS. This paradox indicates that identification of eNOS as a superoxide producer remains elusive. Here, the role of eNOS in superoxide formation from BH4- depleted human coronary artery endothelial cells will be examined by pharmacological approaches using 1,14-bis-isothiourea, 7-nitroindazole or caveolin-1 scaffolding peptide. These agents specifically inhibit oxygen activation by either impeding enzyme activation and/or blocking heme group. In addition, superoxide formation in COS-7 transfected with wild type eNOS and C99A mutant that has a low affinity off BH4 will be examined. These experiments will reveal the interplay between eNOS/BH4 in superoxide formation. In addition, it will be examined the mechanisms by which BH4 is depleted and regenerated in endothelial cells. To this end, the effect of oxidative stress on GTP cyclohydrolase-I activity and GTP feedback regulatory peptide will be examined. Parallel experiments will examine the role of GFRP in the regulation of BH4. Finally, it will be examined whether nitrosative/oxidative stress changes BH4-binding to eNOS to generate an enzyme that produces superoxide by a BH4-insensitive mechanism. This information is likely to contribute in the identification of risk factors and in the design of new therapeutic interventions to ameliorate vascular function in conditions such as hypertension, atherosclerosis and diabetes.